Immune response, depression and fatigue in relation to support ...

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Jun 12, 2007 - Keywords Breast cancer . Support-group intervention . Immunology . NK cells . NK cytotoxicity . Depression. Introduction. Research has ...
Support Care Cancer (2008) 16:57–65 DOI 10.1007/s00520-007-0275-2

ORIGINAL ARTICLE

Immune response, depression and fatigue in relation to support intervention in mammary cancer patients Christina Lindemalm & Fariba Mozaffari & Aniruddha Choudhury & Helena Granstam-Björneklett & Mats Lekander & Bo Nilsson & Marja-Leena Ojutkangas & Anders Österborg & Leif Bergkvist & Håkan Mellstedt

Received: 22 December 2006 / Accepted: 10 May 2007 / Published online: 12 June 2007 # Springer-Verlag 2007

Abstract Goal of work To study the effect of support intervention on immune function in breast cancer patients. Materials and methods Breast cancer patients from an ongoing prospective randomised quality-of -life study were chosen for assaying immune functions in relation to a support-group intervention program running on a residential basis. Twenty-one women received adjuvant-combined radio-chemotherapy (CT-RT) and 20 women radiotherapy (RT). Eleven CT-RT and ten RT patients were randomised to support-group intervention, the rest served as controls. Immune tests for NK cells and NK-cell cytotoxicity, as well as lymphocyte subpopulations and response to antigen were performed before intervention, 2, 6, and 12 months later, in parallel to controls and healthy volunteers (n=11). Depression, anxiety and fatigue were evaluated by the Hospital Anxiety and Depression (HAD) and the Norwegian Fatigue

questionnaire. The density of NK cell receptors and in vitro quantitation of functional NK cytotoxicity against K562 cell line were evaluated. Four-colour flow cytometry was used to detect signal transduction molecules and cytokine expression. T-cell proliferate response to purified protein derivate (PPD) antigen was evaluated. Results No significant immune effect of support intervention could be found. The immune variables were severely disarranged compared to healthy volunteers but showed a statistically significant improvement over time. The majority of patients suffered from fatigue but had low scores for depression and anxiety. Conclusion No effect on immune parameters could be detected from support intervention. The long-lasting immune suppression might override a putative effect of the intervention. Low depression scores may contribute to the absence of a detectable effect.

C. Lindemalm (*) : F. Mozaffari : A. Choudhury : A. Österborg : H. Mellstedt Immune and Gene Therapy Laboratory, Cancer Centre Karolinska, Karolinska University Hospital Solna, 17176 Stockholm, Sweden e-mail: [email protected]

M.-L. Ojutkangas : L. Bergkvist Centre for Clinical Research, Uppsala University, Central Hospital, 72189 Västerås, Sweden

H. Granstam-Björneklett Department of Oncology, Central Hospital, 72189 Västerås, Sweden M. Lekander Department of Clinical Neuroscience, Osher Center for Integrative Medicine and Section of Psychology, Karolinska Institutet, 17177 Stockholm, Sweden B. Nilsson Department for Cancer Epidemiology, Karolinska University Hospital, 17176 Stockholm, Sweden

A. Österborg : H. Mellstedt Departments of Haematology and Oncology, Karolinska University Hospital, Stockholm, Sweden L. Bergkvist Department of Surgery, Central Hospital, 72189 Västerås, Sweden

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Keywords Breast cancer . Support-group intervention . Immunology . NK cells . NK cytotoxicity . Depression

Support Care Cancer (2008) 16:57–65

of depression, anxiety and fatigue in women with primary breast cancer after completed surgery and adjuvant treatment.

Introduction Materials and methods Research has confirmed functional relations between the brain and the immune system and the influence of the immune system on neural, endocrine functions and behaviour [1]. Therefore, behavioural factors may influence biological processes of clinical importance, and peripheral immune or treatment-related events might affect the psychological functioning of a given patient. Many patients facing the diagnosis of cancer react with anxiety and depression [26, 44], and a close relation to immune alterations and depression has been described [9, 40]. Altered NK-cell function and progressive deficiencies in cell-mediated immunity in breast cancer have been noticed many months before overt metastasis [31], and increased numbers of metastases have been related to decreased NK-cell activity both in animal systems and humans [18, 23, 34]. NK-cell counts and sIL-2 levels have also been associated with oestrogen receptor status and age in breast cancer women indicating the importance of the immune system [21, 37]. Different treatments are also related to immune alterations. Surgery alone affects the NK-cell activity in breast cancer women [27], and surgery combined with chemotherapy have an even greater impact on the NK-cell functions [6, 7]. Adjuvant endocrine treatment adds to the complexity [7, 25, 42] as well as stage of the disease, number of chemotherapy courses [28] and timing of assessments [15]. Different tumour treatments also influence cognitive functions and psychological symptoms [2, 28, 45]. Deficits in verbal and visual memory attention and mental flexibility have been described in patients receiving chemotherapy [28]; women with more aggressive chemotherapy experience more side effects [45], and the stage of the disease and the number of courses are of additional importance [28]. Fatigue is another important symptom in cancer patients [24, 32, 41]. Psychological and behavioural stress reduction intervention has been reported to improve psychological symptoms [3, 4, 10, 13, 16, 29] and also certain immune variables [4, 10]. However conflicting results have been reported concerning immune changes in breast cancer after interventions.

Aim The aim of the present study was to analyse the effect of support intervention on immune functions and levels

Forty-one breast cancer patients from an ongoing prospective randomised study examining quality of life issues in relation to support intervention were chosen for assaying immune functions. The first patients in each stratum of treatment were consecutively recruited to the immune study. Twenty-one women received adjuvantcombined radio-chemotherapy (CT-RT) and 20 women radiotherapy (RT). Eleven CT-RT (group A) and ten RT patients (group C) were randomised to support-group intervention; ten CT-RT patients (group B) and ten RT patients (group D) served as controls. All patients were treated in the same surgery and oncology unit, Västerås Hospital, according to Regional Treatment Guidelines. Eligible patients were those who were free from distant metastasis or other complicating co-morbidity, had no earlier malignant disease and were able to fill in forms. Patients were given oral and written information and signed an informed consent. The study was approved by the regional ethics committee. The median time from end of RT to the first immune test was 49 days (range, 5– 97 days) for the intervention group and 31 days (range, 5– 100 days) for the non-intervention patients. Support intervention took place within 4 months after the patients had finished their adjuvant treatment. Post-treatment patients were immune tested according to protocol pre-intervention 2, 6 and 12 months after the first test. In addition, 11 healthy donors were immune tested to exclude laboratory variations over time. They were tested in parallel at time zero and at 2 and 6 months after their first test. To minimise diurnal influences, all blood samples were drawn between 8:00 and 9:00 A.M. Treatment Chemotherapy was mostly given as conventional 5fluorouracil, epirubicin and cychlophosphamide (FEC). Radiation (6 MeV fotones) was delivered to the breast in fractions of 2 Gy doses for a total of 50 Gy to all patients with breast conserving operations (n=36). Patients with lymph node involvement had additional radiation delivered to adjacent lymph nodes. Patients having mastectomy received RT towards the chest wall. Comparable number of patients in both groups received adjuvant tamoxifen therapy. Clinical characteristics of the patients are shown in Table 1.

Support Care Cancer (2008) 16:57–65

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Support-group program The support-intervention program ran from Sunday to Saturday on a residential basis followed by 4 days followup 2 months after the initial visit. The members of the support team were four oncologists, two social workers, two art therapists, two masseuses and a person trained in qigong and mental

visualisation. All personnel had several years of occupational experiences. The guests received information about cancer aetiology, risk factors, treatment, physical and psychological effects and coping mechanisms. Physical exercise, relaxation training, qigong and non-verbal communication (art therapy) were mixed with the theoreticaleducational lectures [22]. Questionnaires

Table 1 Surgical intervention, adjuvant tamoxifen treatment, tumour and clinical characteristics for breast cancer women in intervention group (A=CT+RT and C=RT) and non-intervention group (B=CT+RT and D=RT) Group

Age Surgery Mastectomy Breast conservation Sentinel node biopsy Axillary clearance No axillary dissection Cancer in situ Lymph nodes Negative ≤3 4–8 ≥9 Lgll not done Receptors ER+ ER− Not known PR+ PR− Not known Her 2+ Her 2− Her 2 not known Tumor size ≤2cm >2cm Premenopausal Tamoxifen usage No Tamoxifen Hormone, pre ca diagnosis Married Widowed, single Children at home

A (n=11)

B (n=10)

C (n=10)

D (n=10)

56 (43–69)

69 (55–80)

57 (38–69)

63 (52–69)

1 10

1 9

1 9

2 8

2

4

6

5

9 0

6 0

1 3

4 1

0

0

2

0

1 7 2 1 0

4 4 2 0 0

6 0 1 0 3(2DCIS)

9 0 0 0 1

8 2 1 5 5 1 3 3 5

6 4 0 4 6 0 1 3 6

8 0 2(DCIS) 4 4 2(DCIS) 1 0 9

9 1 0 6 3 1 0 1 9

8 3 3 8 3 0

8 2 2 7 3 3

5 5 0 6 4(2DCIS) 2

9 1 1 5 5 2

11 0 4

8 2 0

9 1 2

10 0 0

The Hospital Anxiety and Depression Scale The Hospital Anxiety and Depression (HAD) Scale [50] was designed to discriminate between anxiety and depression. Seven items add up to an anxiety subscale and seven items to a depression subscale. Each item is rated on a fourpoint scale (0, less than before; 1, not so much; 2, quite a lot; 3, very much) giving a maximum of 21 for depression and anxiety respectively. Scores >10 on either subscale indicate clinical depression or anxiety whilst subscale scores in the range of 8–10 represent borderline cases. The Norwegian Fatigue Questionnaire The Norwegian Fatigue Questionnaire, the Norwegian version of The Fatigue Formula (FQ) [11], accessed fatigue. It was originally validated in primary care and lymphoma patients [24, 32]. It is a self-reporting instrument consisting of four response alternatives to 11 symptoms experienced during the last period of time compared to how the patient felt when last feeling well. The responses are summed up to two subscales, physical fatigue (PF) and mental fatigue (MF), and all items are designated total fatigue (TF) [24, 32]. Immune analyses Monoclonal antibodies and other reagents Antibodies conjugated with flourescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) against the surface molecules CD3, CD4, CD8, CD25, CD28, CD56, CD161 and NKB1 and the cytokines interferon gamma (IFN-γ), interleukin 2 (IL-2) and IL-4, as well as isotype-matched negative controls were commercially purchased from Becton-Dickinson (BD), Mountain view, CA, USA. Mabs against the signal transduction molecules P56lck, p59fyn Zap70 and PI3k were purchased from Transduction Laboratories (Lexington, KY, USA) and antibody against CD3ζ from Bio Site, Stockholm, Sweden. Saponin, PMA and ionomycin were purchased from Sigma (St Louis, MO, USA), and Brefeldin A and goat anti-mouse Mab were obtained from BD.

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Isolation of peripheral blood mononuclear cells and cell cultures Peripheral blood mononuclear cells (PBMNC) were isolated from heparinized blood by separation on a FicollIsopaque gradient (Amersham Pharmacia Biotech AB, Uppsala Sweden). To assess the ability of T and NK cells to produce cytokines in response to stimuli, 1–2×106 cells/ ml were stimulated with 25 ng/ml paramethoxyamphetamine (PMA) and 1 μg/ml ionomycin in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal calf serum (FCF), 2 mM/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all reagents (Gibco BRL, Paisley, UK), 10 μg/ml Brefeldin A in Falcon tubes. Unstimulated samples were set up in parallel without PMA and ionomicin. The tubes were incubated at 37°C in 5% CO2 for 4 h.

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Proliferative assay Mononuclear cells from patients or healthy volunteers (1× 105 cells/well) were incubated in medium alone, medium containing 10 μg/ml phytohaemagglutinin (PHA; Gibco BRL) or 2.5 μg/ml of mycobacterial-purified protein derivative (PPD; National Serum Institute, Copenhagen, Denmark) in a 96-well culture plate. Cultures were incubated for 3 days and 1 μCi/well 3H-thmidine (Amersham Pharmacia Biotech) was added to each well for the final 16–18 h. Cells were harvested, and the incorporated radioactivity was measured in a β-counter (Micro Beta 1450, Wallace, Turku, Finland). Results were reported as stimulation index calculated as the ratio of radioactivity of cells incubated with PHA or PPD and the radioactivity of control cultures. Statistics

Cellular staining and flow cytometry Flow-cytometric analyses were carried out using a FACS Calibur (BD) as described earlier [24].

Descriptive measures (mean±SD) are reported. Repeated data within groups were analysed with repeated measurement analysis of variance (ANOVA). A p value≤0.05 was considered to indicate statistical significance.

Calculation of absolute cell numbers Lymphocytes expressing NK receptors and T-cell subset markers as determined by flow cytometry were expressed as percentage of the total population. To determine the number of the populations per millilitre of blood, the percentage fraction of lymphocytes was multiplied by the number of lymphocytes per litre as determined by an automated differential blood count on the same sample. NK-cell-mediated cytotoxicity NK-cell function was measured in vitro using a chromium release assay. Briefly, NK-sensitive K562 cells were labelled with 100 μCi Na251CrO4 (37 MBq, 1mCi, Amersham, UK) for 1 h and co-cultured with effector cells for 4 h at various effector: target cell ratios in triplicates in 96-well plates (50:1, 25:1, 12.5:1, 6.25:1). Spontaneous 51 Cr release was determined by incubating target cells alone and total release by lysing labelled cells with 5% Triton X-100. After incubation, the supernatant was counted in a gamma counter. NK cytotoxic activity was calculated as number of lytic units (LU) per 106 effector cells. One LU is the number of effector cells capable of lysing 30% of the target cells, and the calculations were performed using a computer software kindly provided by Dr. T. Whiteside, University of Pittsburgh, Pittsburgh, PA.

Results The HAD questionnaire Anxiety score More patients in the support group had higher anxiety scores (5/21, 24% had scores >10) at the start of the intervention compared to the non-support group (0/20). The mean value for anxiety in the support group was also higher compared to the non support group and improved significantly over time (p